共查询到20条相似文献,搜索用时 31 毫秒
1.
Channels of maize starch granules are lined with proteins and phospholipids. Therefore, when they are treated with reagents that react at or near the surfaces of channels, three types of crosslinks could be produced: protein–protein, protein–starch, starch–starch. To determine which of these may be occurring and the effect(s) of channel proteins (and their removal) on crosslinking, normal and waxy maize starches were treated with a proteinase (thermolysin, which is known to remove protein from channels) before and after crosslinking, and the properties of the products were compared to those of a control (crosslinking without proteinase treatment). After establishing that treatment of starch with thermolysin alone had no effect on the RVA trace, three reaction sequences were used: crosslinking alone (CL), proteinase treatment before crosslinking (Enz-CL), proteinase treatment after crosslinking (CL-Enz). Two crosslinking reagents were used: phosphoryl chloride (POCl3), which is known to react at or near channel surfaces; STMP, which is believed to react throughout the granule matrix. Three concentrations of POCl3 (based on the weight of starch) were used. For both normal maize starch (NMS) and waxy maize starch (WMS) reacted with POCl3, the trends were generally the same, with apparent relative degrees of crosslinking indicated to be CL-Enz = CL > Enz-CL, but the effects were greater with NMS and there were differences when different concentrations of reagent were used. The basic trends were the same when potato starch was used in the same experiments. Crosslinking with STMP was done both in the presence and the absence of sodium sulfate (SS). Both with and without SS and with both NMS and WMS, the order of indicated crosslinking was generally the same as found after reaction with POCl3, with the indicated swelling inhibition being greater when SS was present in the reaction mixture. Examination of the maize starches with a protein stain indicated that channel protein was removed by treatment with thermolysin when the proteinase treatment occurred before crosslinking with either POCl3 or STMP, but only incompletely or not at all if the treatment with the proteinase occurred after crosslinking. Because the crosslinking reactions were less effective when the protein was removed, the results are tentatively interpreted as indicating that they involved protein molecules, although there may not be a direct relationship. 相似文献
2.
Starch
granules from maize (Zea mays) contain a characteristic
group of polypeptides that are tightly associated with the starch
matrix (C. Mu-Forster, R. Huang, J.R. Powers, R.W. Harriman, M. Knight,
G.W. Singletary, P.L. Keeling, B.P. Wasserman [1996] Plant Physiol
111: 821–829). Zeins comprise about 50% of the granule-associated
proteins, and in this study their spatial distribution within the
starch granule was determined. Proteolysis of starch granules at
subgelatinization temperatures using the thermophilic protease
thermolysin led to selective removal of the zeins, whereas
granule-associated proteins of 32 kD or above, including the waxy
protein, starch synthase I, and starch-branching enzyme IIb, remained
refractory to proteolysis. Granule-associated proteins from maize are
therefore composed of two distinct classes, the surface-localized zeins
of 10 to 27 kD and the granule-intrinsic proteins of 32 kD or higher.
The origin of surface-localized δ-zein was probed by comparing
δ-zein levels of starch granules obtained from homogenized whole
endosperm with granules isolated from amyloplasts. Starch granules from
amyloplasts contained markedly lower levels of δ-zein relative to
granules prepared from whole endosperm, thus indicating that δ-zein
adheres to granule surfaces after disruption of the amyloplast
envelope. Cross-linking experiments show that the zeins are deposited
on the granule surface as aggregates. In contrast, the
granule-intrinsic proteins are prone to covalent modification, but do
not form intermolecular cross-links. We conclude that individual
granule intrinsic proteins exist as monomers and are not deposited in
the form of multimeric clusters within the starch matrix.It has long been known that starch granules contain bound
polypeptides, with protein levels of isolated starch granules from
maize (Zea mays) ranging from 0.3 to 1.0% based upon
measurement of N2 (May, 1987). A recent study by our
laboratory demonstrates that isolated starch granules from maize
contain several dozen strongly bound polypeptides (Mu-Forster et al.,
1996). The granule-associated proteins include starch-biosynthetic
enzymes such as the waxy protein, SSI, and SBEIIb. These polypeptides
are not removed from intact starch granules by protease treatment or
detergent washing; therefore, they are believed to bind to the starch
and to become irreversibly entrapped within the starch
matrix.Based upon staining intensities of polypeptides extracted from the
starch granule (Mu-Forster et al., 1996), approximately one-half of the
granule-associated proteins in maize consist of low-molecular-mass
polypeptides ranging between 10 and 27 kD. These bands fall within the
size range displayed by the zein storage proteins, however, the spatial
distribution of these polypeptides within the starch granule is
unknown. Zeins have been defined as alcohol-soluble proteins that occur
principally in protein bodies of maize endosperm and that may or may
not require reduction before extraction (Wilson, 1991). The association
of zeins with starch granules during endosperm development would not be
expected because zein genes do not contain transit peptides that would
target these proteins through the amyloplast envelope into the
amyloplast stroma.The objective of this study was to establish the topology of
granule-associated zeins in starch granules from maize endosperm. To
accomplish this, it was necessary to distinguish between
surface-localized and internalized polypeptides. Our working hypothesis
defines polypeptides localized at the starch granule surface as those
that are susceptible to hydrolysis upon treatment of intact granules
with exogenous proteases. Conversely, internal granule proteins are
defined as those that (a) become susceptible to proteolysis only
following thermal disruption of the starch matrix, and (b) resist
extraction by 2% SDS at room temperatures (Denyer et al., 1993; Rahman
et al., 1995; Mu-Forster et al., 1996).In this study we were able to distinguish between surface-localized and
internalized granule-associated polypeptides in starch granules
from maize endosperm by use of the thermophilic protease thermolysin.
Thermolysin is well suited for this purpose because it is highly active
at starch-gelatinization temperatures, and has also been shown to
effectively hydrolyze hydrophobic proteins located at the surfaces of
chloroplasts and other subcellular organelles (Cline et al., 1984; Xu
and Chitnis, 1995). Upon extended incubation of intact starch granules
with thermolysin at subgelatinization temperatures, we found that zeins
were selectively removed from the starch granule surface. All other
granule-associated polypeptides remained inaccessible to proteolytic
attack or to extraction by 2% SDS, unless the starch matrix was first
disrupted by gelatinization. Our results distinguish between the
surface-localized and granule-intrinsic proteins of maize endosperm,
and establish that zeins are localized at the starch-granule surface.
In addition, cross-linking experiments were conducted to determine
nearest-neighbor relationships among zein subunits localized at the
granule surface and granule intrinsic polypeptides localized within the
starch matrix. 相似文献
3.
Differential effects of Wx-A1, -B1 and -D1 protein deficiencies on apparent amylose content and starch pasting properties in common wheat 总被引:27,自引:0,他引:27
M. Yamamori N. T. Quynh 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》2000,100(1):32-38
Waxy (Wx) protein is a granule-bound starch synthase (GBSS) responsible for amylose production in cereal endosperm. Eight
isolines of wheat (Triticum aestivum L.) having different combinations of presence and absence of three Wx proteins, Wx-A1, -B1, and -D1, were produced in order
to elucidate the effect of Wx protein deficiencies on the apparent amylose content and starch-pasting properties. An improved
SDS gel electrophoresis showed that ’Bai Huo’ (a parental wheat) carried a variant Wx-B1 protein from an allele, Wx-B1e. Thus, wheat lines of types 1, 2, 4, and 6 examined in this study contained a variant Wx-B1 allele and not the standard allele, Wx-B1a. The results from 3 years of experiments using 176 lines derived from two cross-combinations showed that apparent amylose
content increased the least in type 8 (waxy) having no Wx proteins and, in ascending order, increased in type 5 (only the
Wx-A1 protein is present) <type 7 (Wx-D1) <type 6 (Wx-B1) <type 3 (Wx-A1 and -D1) <type 4 (Wx-A1 and -B1) <type 2 (Wx-B1 and
-D1) <type 1 (three Wx proteins). However, Tukey’ s studentized range test did not detect significant differences in some
cases. Densitometric analysis suggested that the amylose content was related to the amount of the Wx protein in the eight
types. Parameters in the Rapid Visco-Analyzer test and swelling power were correlated to amylose content. Consequently, amylose
content and pasting properties of starch were determined to be influenced the most by the lack of the Wx-B1 protein, followed
by a lack of Wx-D1, and leastly by the Wx-A1 deficiency, which indicated the presence of differential effects of the three
null alleles for the Wx protein.
Received: 1 February 1999 / Accepted: 10 April 1999 相似文献
4.
Xiaoping Chen Zhangying Wang Jianhua Wang Maoyan Wang Li Zhao Guoying Wang 《Plant Cell, Tissue and Organ Culture》2007,88(1):11-20
ADP-glucose pyrophosphorylase (AGPase) represents a key regulatory step in starch synthesis. A 0.9 kb of 5′ flanking region
preceding Brittle2 gene, encoding the small subunit of maize endosperm AGPase, was cloned from maize genome and its expression pattern was studied
via the expression of β-glucuronidase (GUS) gene in transgenic tobacco. Analysis of GUS activities showed that the 0.9 kb
fragment flanking Brittle2 gene was sufficient for driving the seed-preferred expression of the reporter gene. The activity of the 0.9 kb 5′ flanking
fragment was compared with that of the tandem promoter region from a zein gene (zE19, encoding a maize 19 kDa zein protein). The results indicated that both promoters were seed-preferred in a dicotyledonous
system as tobacco and the activity of zE19 promoter was three to fourfold higher than that of the 0.9 kb fragment flanking Brittle2 gene in transgenic tobacco seeds. At the same time, zE19-driven GUS gene expressed earlier than Brittle2 promoter during seed development. Histochemical location of GUS activity indicated that both promoters showed high expression
in embryos, which is different from similar promoters tested in maize. 相似文献
5.
Protein accumulation and rumen stability of wheat γ‐gliadin fusion proteins in tobacco and alfalfa 下载免费PDF全文
Xiaodong Sun Cecilia L. Chi‐Ham Tamar Cohen‐Davidyan Christopher DeBen Girma Getachow Edward DePeters Daniel Putnam Alan Bennett 《Plant biotechnology journal》2015,13(7):974-982
The nutritional value of various crops can be improved by engineering plants to produce high levels of proteins. For example, because methionine deficiency limits the protein quality of Medicago Sativa (alfalfa) forage, producing alfalfa plants that accumulate high levels of a methionine‐rich protein could increase the nutritional value of that crop. We used three strategies in designing methionine‐rich recombinant proteins that could accumulate to high levels in plants and thereby serve as candidates for improving the protein quality of alfalfa forage. In tobacco, two fusion proteins, γ‐gliadin‐δ‐zein and γ‐δ‐zein, as well as δ‐zein co‐expressed with β‐zein, all formed protein bodies. However, the γ‐gliadin‐δ‐zein fusion protein accumulated to the highest level, representing up to 1.5% of total soluble protein (TSP) in one transformant. In alfalfa, γ‐gliadin‐δ‐zein accumulated to 0.2% of TSP, and in an in vitro rumen digestion assay, γ‐gliadin‐δ‐zein was more resistant to microbial degradation than Rubisco. Additionally, although it did not form protein bodies, a γ‐gliadin‐GFP fusion protein accumulated to much higher levels, 7% of TSP, than a recombinant protein comprised of an ER localization signal fused to GFP in tobacco. Based on our results, we conclude that γ‐gliadin‐δ‐zein is a potential candidate protein to use for enhancing methionine levels in plants and for improving rumen stability of forage protein. γ‐gliadin fusion proteins may provide a general platform for increasing the accumulation of recombinant proteins in transgenic plants. 相似文献
6.
For the production of α-D-glucose-1-phosphate (G-1-P), α-1,4-D-glucan phosphorylase from Thermus caldophilus GK24 was partially purified to a specific activity of 13 U mg−1 and an enzyme recovery of 15%. The amount of G-1-P reached maximum (18%) when soluble starch was used as substrate, and the
smallest substrate for G-1-P formation was maltotriose. The structure of purified G-1-P was confirmed by comparison to 13C-NMR data for an authentic sample. In addition to G-1-P, glucose-6-phosphate (12%) was simultaneously produced when 10 mM
maltoheptaose was used as substrate. Journal of Industrial Microbiology & Biotechnology (2000) 24, 89–93.
Received 12 May 1999/ Accepted in revised form 29 August 1999 相似文献
7.
Despite the well-characterized function of the green-algal eyespot apparatus as a combined absorption/reflection screen for
the photoreceptor for phototaxis, little is known about the proteins involved in the formation of this complex organelle.
We therefore purified the carotenoid-rich lipid globules, which are the most conspicuous component of the eyespot sensu strictu
from Spermatozopsis similis Preisig et Melkonian. Electron microscopy and an average carotenoid:chlorophyll ratio of 51, confirmed the high purity of
the fraction. The diameter of isolated globules (approx. 112 nm) fell within their in vivo range (90–120 nm). Absorption spectra
in aqueous media peaked at 535 nm. The predominant carotenoids were β,ψ-, β, β- and δ-carotene. Freeze-fracture studies with
cells and whole-mount electron microscopy of isolated globules demonstrated regularly arranged particles at the globule surface.
Sodium dodecyl sulfate–polyacrylamide gel electrophresis revealed specific enrichment of 10 tightly bound major proteins and
several minor proteins with the globules. Proteases were used to analyze their topology and function. Upon treatment with
thermolysin, globules were released from a fraction enriched in isolated eyespot apparatuses. Major proteins of these globules,
and those treated with thermolysin after isolation, were identical. However, the purified proteins were sensitive to thermolysin,
indicating that domains of them are normally hidden in the globule matrix. In contrast, pronase degraded all globule-associated
proteins in situ. These globules were not stable and easily fused, whereas thermolysin-treated globules were relatively stable.
Lipase did not affect globule stability. These results indicate that the five thermolysin-resistant proteins (apparent M
r values: 56, 52, 32, 29, 27 kDa) are close to the surface and might be crucial for globule stabilization, whereas the thermolysin-accessible
proteins are probably involved in globule/globule interactions and/or globule/eyespot-membrane interactions.
Received: 19 June 2000 / Accepted: 4 October 2000 相似文献
8.
Päivi L. H. Rinne Päivi L. M. Kaikuranta Linus H. W. van der Plas Chris van der Schoot 《Planta》1999,209(4):377-388
Dehydrins accumulate in various plant tissues during dehydration. Their physiological role is not well understood, but it
is commonly assumed that they assist cells in tolerating dehydration. Since in perennials the ability of the shoot apex to
withstand dehydration is pivotal for survival through winter, we investigated if and how dehydrins may be involved. A first
step in assessing such a role is the identification of their subcellular location. We therefore mapped the location of dehydrin
homologues, abscisic acid-responsive (RAB 16-like) polypeptides, in the apex of birch (Betula pubescens Ehrh.). In non-cold-acclimated plants a single low-abundant RAB 16-member (a 33-kDa polypeptide) was produced, and localized
in the cytoplasm only. During cold acclimation two additional members were produced (24 and 30 kDa) and accumulated in nuclei,
storage protein bodies and starch-rich amyloplasts. Western blots of proteins isolated from purified starch granules and from
protein bodies revealed the presence of the 24-kDa dehydrin. Since starch and protein reserves are gradually consumed during
winter, serving cell maintenance, starch- and protein-degrading enzymes must remain locally active. We therefore investigated
the hypothesis that dehydrins might create local pools of water in otherwise dehydrated cells, thereby maintaining enzyme
function. In agreement with our hypothesis, enzyme assays showed that under conditions of low water activity a partially purified
dehydrin fraction was able to improve the activity of α-amylase (EC 3.2.1.1.) relative to fractions from which dehydrin was
removed by immunoprecipitation. The results confirm the general belief that dehydrins serve desiccation tolerance, and suggest
that a major function is to rescue the metabolic processes that are required for survival and re-growth.
Received: 12 September 1998 / Accepted: 19 April 1999 相似文献
9.
Federica Taddei Laura Gazza Salvatore Conti Vera Muccilli Salvatore Foti Norberto Edgar Pogna 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》2009,119(7):1205-1212
The starch granule proteins from 113 einkorn wheat (Triticum monococcum ssp monococcum) accessions were analyzed by acidic, polyacrylamide gel electrophoresis (A-PAGE), and two-dimensional A-PAGE x SDS-PAGE.
All accessions were confirmed to contain equal amounts of two polypeptide chains corresponding to puroindoline B (Pin-B),
as well as a prominent component plus a faint band corresponding to puroindoline A (Pin-A). When compared with soft-textured
common wheat, “monococcum” accessions showed an increase of 3.2- and 2.7-fold in Pin-A and Pin-B levels on the starch granules,
respectively. In addition, all accessions contained a novel component of the 2S super-family of seed proteins named Einkorn
Trypsin Inhibitor (ETI), which was found to be encoded as a pre-protein 148 residues long. Wild-type ETI encoded by allele
Eti-A
m
1a and “valine-type” ETI encoded by allele Eti-A
m
1b, which occurred in 107 and six einkorn accessions, respectively, were found to accumulate on starch granules as a mature
protein of 121 amino acids with a hydrophobic central domain. The einkorn accessions exhibited an average SKCS index as low
as −2.05 ± 11.4, which is typical of extra-soft kernels. The total surface area of starch granules in “monococcum” wheat,
as determined by visual assessments in counting chambers, was estimated at 764 mm2/mg of starch, and was about 1.5 times higher than that for common wheat. The results are discussed in relation to the identification
of factors that cause the extra-soft texture of einkorn kernels. 相似文献
10.
Cabantous S Pédelacq JD Mark BL Naranjo C Terwilliger TC Waldo GS 《Journal of structural and functional genomics》2005,6(2-3):113-119
We have improved our green fluorescent protein (GFP) folding reporter technology [Waldo et al., (1999) Nat. Biotechnol. 17, 691–695] to evolve recalcitrant proteins from Mycobacterium tuberculosis. The target protein is inserted into the scaffolding of the GFP, eliminating false-positive artifacts caused by expression
of truncated protein variants from internal cryptic ribosome binding sites in the target RNA. In parallel, we have developed
a new quantitative fluorescent protein tagging and detection system based on micro-domains of GFP. This split-GFP system,
which works both in vivo and in vitro, is amenable to high-throughput assays of protein expression and solubility [Cabantous et al., (2005) Nat. Biotechnol. 23, 102–107]. Together, the GFP folding reporter and split-GFP technologies offer a comprehensive system for manipulating and
improving protein folding and solubility. 相似文献
11.
Previous studies from this laboratory have shown that the thermolysin fragment 121–316, comprising entirely the“all-α” COOH-terminal
structural domain 158–316, as well as fragment 206–316 (fragment FII) are able to refold into a native-like, stable structure
independently from the rest of the protein molecule. The present report describes conformational properties of fragments 228–316
and 255–316 obtained by chemical and enzymatic cleavage of fragment FII, respectively. These subfragments are able to acquire
a stable conformation of native-like characteristics, as judged by quantitative analysis of secondary structure from far-ultra-violet
circular dichroism spectra and immunochemical properties using rabbit anti-thermolysin antibodies. Melting curves of the secondary
structure of the fragments show cooperativity with a temperature of half-denaturationT
mof 65–66°C. The results of this study provide evidence that it is possible to isolate stable supersecondary structures (folding
units) of globular proteins and correlate well with predictions of subdomains of the COOH-terminal structural domain 158–316
of thermolysin. 相似文献
12.
Mutations that reduced the rate of starch synthesis in pea (Pisum sativum L.) embryos through effects on enzymes on the pathway from sucrose to adenosine 5′-diphosphoglucose (ADPglucose) also led
to a reduction in the amylose content of the starch of developing embryos. Evidence is presented that this relationship between
rate of synthesis and the composition of starch is due to the fact that amylopectin-synthesising isoforms of starch synthase
have higher affinities for ADPglucose than the amylose-synthesising isoform. First, developing mutant embryos (rb, rug3 and rug4 mutants) displayed both reduced amylose contents in their starches and reduced ADPglucose contents relative to wild-type
embryos. Second, incubation of detached, wild-type embryos for 6 h at high and low glucose concentrations resulted in differences
in both ADPglucose content and the relative rates of amylose and amylopectin synthesis. At 0.25 M glucose both ADPglucose
content and the proportion of synthesised starch that was amylose were about twice as great as at 25 μM glucose. Third, S
0.5 values for soluble (amylopectin-synthesising) starch synthases in developing embryos were several-fold lower than that for
granule-bound (amylose synthesising) starch synthase. Estimates of the expected amylose contents of the starch of the mutant
embryos, based on the reduction in their ADPglucose contents and on the S
0.5 values of the starch synthases, were very similar to the measured amylose contents. The implications of these results for
the determination of starch composition are discussed.
Received: 6 February 1999 / Accepted: 22 May 1999 相似文献
13.
Raw starch-digesting amylases (RSDAs) in many microorganisms convert starch granules into maltodextrins and simple sugars.
We cloned and sequenced from Cytophaga sp. an RSDA with an excellent raw starch digestion activity. This RSDA was highly inducible by raw starch, but not by other
sugars, suggesting that an unknown signal transduction mechanism is involved in the degradation of raw starch. We used a proteomic
approach to investigate the effect of raw starch on protein expression in Cytophaga sp. Using MALDI–TOF MS protein analysis, we have identified three proteins up-regulated by raw starch, i.e., a 60-kDa chaperonin
(cpn60), glutaminase, and pyruvate phosphate dikinase (PPDK). Subsequent time-course studies detected an increased expression
of RSDA as well as the highest expression of PPDK occurring 6 h post-incubation with raw corn starch, implying that the latter
enzyme may work along with RSDA on the digestion of raw starch. Finding these proteins up-regulated by raw starch may provide
an insight into how Cytophaga sp. cells respond to raw starch stimulation. 相似文献
14.
Pi Nyvall Jerome Pelloux Howard V. Davies Marianne Pedersén Roberto Viola 《Planta》1999,209(1):143-152
Red algae (Rhodophyceae) are photosynthetic eukaryotes that accumulate starch granules in the cytosol. Starch synthase activity
in crude extracts of Gracilaria tenuistipitata Chang et Xia was almost 9-fold higher with UDP[U-14C]glucose than with ADP[U-14C]glucose. The activity with UDP[U-14C]glucose was sensitive to proteolytic and oxidative inhibition during extraction whilst the activity with ADP[U-14C]glucose appeared unaffected. This indicates the presence of separate starch synthases with different substrate specificities
in G. tenuistipitata. The UDPglucose: starch synthase was purified and characterised. The enzyme appears to be a homotetramer with a native Mr of 580 kDa and displays kinetic properties similar to other α-glucan synthases such as stimulation by citrate, product (UDP)
inhibition and broad primer specificity. We propose that this enzyme is involved in cytosolic starch synthesis in red algae
and thus is the first starch synthase described that utilises UDPglucose in vivo. The biochemical implications of the different
compartmentalisation of starch synthesis in red algae and green algae/plants are also discussed.
Received: 29 January 1999 / Accepted: 11 March 1999 相似文献
15.
Freeze-dried, alginate-based beads, used for the immobilization of a denitrifying bacterium (Pseudomonas sp.), were filled with different concentrations (10%, 20%, 30% and 40%, w/w) of granular starch. The beads were incubated
under denitrifying conditions in laboratory-scale, flow-through columns and monitored for changes in their physical and denitrifying
properties. Freeze-dried beads containing high concentrations of starch were found to have better mechanical and denitrifying
properties than beads containing low concentrations of this filler. Nitrate removal by the beads was found to be correlated
with their starch content. Nitrite accumulation, as a result of incomplete denitrification, increased with the decrease in
starch content of the beads. Nitrite in the outlet of the columns was measured in all types of beads during the initial phase
of incubation but was undetectable, with the exception of beads with the lowest starch content, at later stages of incubation.
Received: 9 November 1998 / Received revision: 3 February 1999 / Accepted: 5 February 1999 相似文献
16.
B H Chung Y J Choi S H Yoon S Y Lee Y I Lee 《Journal of industrial microbiology & biotechnology》2000,24(2):94-99
Fed-batch cultures were carried out to overproduce human insulin-like growth factor I (IGF-I) in Escherichia coli. The effects of carbon sources (glucose or glycerol) and induction time on cell growth and IGF-I production were investigated
in more detail. Glycerol was a better carbon source than glucose for IGF-I production in fed-batch culture. Induction at the
mid-exponential phase with glycerol as a carbon source in the pH-stat fed-batch culture was optimal for IGF-I production.
Under this condition, 2.8 g L−1 of fusion IGF-I was produced as inclusion bodies. We have also developed downstream processing for preparative scale purification
of IGF-I from the fusion protein produced by the fed-batch culture using glycerol as a carbon source. After the fusion protein
expressed was solubilized in 8 M urea and cleaved with hydroxylamine, the released IGF-I was purified by cation exchange chromatography,
refolding and preparative scale reverse phase HPLC (rp-HPLC) to give recombinant IGF-I of >98% purity. The biological activities
of the purified IGF-I were measured and found to be identical to those of commercial IGF-I. Journal of Industrial Microbiology & Biotechnology (2000) 24, 94–99.
Received 13 January 1999/ Accepted in revised form 02 October 1999 相似文献
17.
Maize endosperms accumulate during development a large amount of storage proteins (zeins). The rate of zein accumulation is under the control of several regulatory genes. Two of these, the opaque-2 and opaque-6 mutants, lower the zein level, thus improving the nutritional quality of maize meals. An endosperm protein of Mr 32 000 (b-32) appears to be correlated with the zein level. The b-32 protein is encoded by the opaque-6 gene which, in turn, is activated by opaque-2. We report the purification, amino-acid composition and peptide map of b-32 protein. Furthermore we demonstrate that the protein exists as a monomer likely located in the soluble cytoplasm. As a step towards the isolation of a complementary-DNA clone for b-32 protein, the purification of its corresponding mRNA is described.Abbreviations b-32
endosperm protein of Mr 32000
- cDNA
complementary DNA
- EDTA
ethylenediaminetetraacetic acid
-
O2, O6
opaque 2, opaque-6 genes
- PMSF
phenylmethylsulfonylfluoride
- RSP
reduced soluble proteins
- SDS-PAGE
sodium dodecyl sulfate-polyacrylamide gel electrophoresis 相似文献
18.
Use of the green fluorescent protein as an educational tool 总被引:1,自引:0,他引:1
The green fluorescent protein (GFP) is a bioluminescent protein that can be expressed and easily detected as a fully fluorescent
protein in both bacterial and eukaryotic cells. These properties, along with its ability to withstand exposure to denaturants,
organic solvents, high temperature and a wide pH range, make GFP an ideal educational tool. To that end, two GFP-based laboratory
modules are described that can be used to teach recombinant DNA and protein purification techniques to high school and undergraduate
college students. Journal of Industrial Microbiology & Biotechnology (2000) 24, 323–326.
Received 02 April 1999/ Accepted in revised form 20 November 1999 相似文献
19.
Zeins are alcohol soluble seed storage proteins synthesized within the endosperm of maize and subsequently deposited into endoplasmic reticulum (ER) derived protein bodies. The genes encoding the beta and delta zeins were previously introduced into tobacco with the expectation of improving the nutritional quality of plants (Bagga et al. in Plant Physiol 107:13, 1997). Novel protein bodies are produced in the leaves of transgenic plants accumulating the beta or delta zein proteins. The mechanism of protein body formation within leaves is unknown. It is also unknown how zeins are retained in the ER since they do not contain known ER retention motifs. Retention may be due to an interaction of zeins with an ER chaperone such as binding luminal protein (BiP). We have demonstrated protein–protein interactions with the delta zeins, beta zeins, and BiP proteins using an E. coli two-hybrid system. In this study, four putative BiP binding motifs were identified within the delta zein protein using a BiP scoring program (Blond-Elguindi et al. in Cell 75:717, 1993). These putative binding motifs were mutated and their effects on protein interactions were analyzed in both a prokaryotic two-hybrid system and in plants. These mutations resulted in reduced BiP–zein protein interaction and also altered zein–zein interactions. Our results indicate that specific motifs are necessary for BiP–delta zein protein interactions and that there are specific motifs which are necessary for zein–zein interactions. Furthermore, our data demonstrates that zein proteins must be able to interact with BiP and zeins for their stability and ability to form protein bodies. 相似文献
20.
Weenink XO Punt PJ van den Hondel CA Ram AF 《Applied microbiology and biotechnology》2006,69(6):711-717
Although filamentous fungi have a unique property of secreting a large amount of homologous extracellular proteins, the use
of filamentous fungi as hosts for the production of heterologous proteins is limited because of the low production levels
that are generally reached. Here, we report a general screening method for the isolation of mutants with increased protein
production levels. The screening method makes use of an Aspergillus niger strain that lacks the two major amylolytic enzymes, glucoamylase (GlaA) and acid amylase (AamA). The double-mutant strain
grows poorly on starch and its growth is restored after reintroducing the catalytic part of the glucoamylase gene (GlaA512). We show that the fusion of a heterologous protein, a laccase from Pleurotus ostreatus (Pox2), to the catalytic part of glucoamylase (GlaA512–Pox2) severely hampers efficient production of the glucoamylase protein, resulting in a slow-growth phenotype on starch.
Laccase-hypersecreting mutants were obtained by isolating mutants that displayed improved growth on starch plates. The mutant
with the highest growth rate on starch displayed the highest laccase activity, indicating that increased glucoamylase protein
levels are correlated with higher laccase production levels. In principle, our method can be applied to any low-produced heterologous
protein that is secreted as a fusion with the glucoamylase protein. 相似文献